JP7486015B2 - Component for genetic testing container, and genetic testing container - Google Patents

Description

The present invention relates to genetic testing technology, and in particular to the simplification of the reaction solution preparation process for amplifying nucleic acids.

Conventionally, when preparing a reaction solution for amplifying nucleic acid in genetic testing, the user generally had to prepare minute amounts of solution (a few μL to a few tens of μL) multiple times using a genetic testing container such as a tube.
Specifically, for example, when preparing a reaction solution for PCR, a process was required in which four types of reagents, namely a buffer solution, a substrate (nucleic acid synthesis substrate), a primer, and an enzyme (nucleic acid synthesis enzyme), were injected into a tube to prepare the reaction solution, as shown in FIG.
In other words, the user had to perform preparation four times per sample and then inject the nucleic acid to be tested (hereinafter, sometimes referred to as the target nucleic acid) into the tube. Since preparation of such a small amount of solution is very complicated, it would be beneficial if the process could be simplified.

One possible method for solving such problems is to mix various reagents and freeze-dry them to prepare a mixture, which can then be used (see Patent Document 1).
That is, if the manufacturer provides a tube with a freeze-dried mixture of buffer, substrate, primer, and enzyme pre-fixed inside, the user can eliminate four preparation steps.

Patent No. 4468979

However, this type of freeze-drying method requires the preparation of equipment such as a vacuum dryer. In addition, when performing vacuum drying, it is essential to carry out the process in a low-humidity environment to prevent the enzymes from becoming unstable or inactivated due to moisture, and special facilities are required.

The inventors therefore conducted extensive research into a method that can simplify the preparation process of a reaction solution for amplifying nucleic acids without using special equipment or facilities, and succeeded in simplifying the preparation process by mixing various reagents with a resin composition and producing a genetic testing container component and a genetic testing container in which this mixture is fixed, thereby completing the present invention.

In other words, the present invention aims to provide a component for a genetic testing container and a genetic testing container that can simplify the process of preparing a reaction solution for amplifying DNA without requiring special equipment or facilities.

To achieve the above object, the genetic testing container member of the present invention is configured such that a mixture containing one or more gene amplification reagents, including at least a nucleic acid synthesizing enzyme, and a resin composition is applied to at least a portion of a substrate.

In addition, it is preferable that the genetic testing container member of the present invention is configured such that the resin composition is a photosensitive resin composition and the surface of the mixture is cured by irradiation with ultraviolet light.

In addition, it is preferable that the gene amplification reagent of the genetic testing container member of the present invention contains at least one selected from the group consisting of a nucleic acid synthesis substrate, a primer, and a buffer solution together with a nucleic acid synthesis enzyme.

It is also preferable that the genetic testing container member of the present invention is configured so that the genetic testing is performed using an amplified product obtained by the PCR method.

Furthermore, the genetic testing container of the present invention is formed using any of the genetic testing container members described above, and the mixture is fixed within the container.

The present invention makes it possible to provide a component for a genetic testing container and a genetic testing container that can simplify the process of preparing a reaction solution for amplifying nucleic acids without requiring special equipment or facilities.

FIG. 1 shows the results of amplification of target nucleic acids in an example. FIG. 1 shows a general method for preparing a reaction solution for PCR and a method for preparing a reaction solution for PCR using a freeze-dried mixture.

Hereinafter, an embodiment of the member for a genetic testing container and the genetic testing container of the present invention will be described in detail. However, the present invention is not limited to the specific contents of the following embodiment and the examples described later.
The member for a genetic testing container of this embodiment is characterized in that a mixture containing one or more gene amplification reagents containing at least a nucleic acid synthesizing enzyme and a resin composition is applied to at least a part of a substrate.

In this embodiment, the genetic testing container member is a member for mainly manufacturing the genetic testing container, and a film or sheet shape can be suitably used. The genetic testing container member of this embodiment can also be used as a packaging material or package.

As the material for the genetic testing container member of this embodiment, polyolefin resins such as polyethylene and polypropylene can be suitably used. For example, polyethylene, copolymers of ethylene and α-olefin, copolymers of ethylene and vinyl acetate, ionomers using ethylene, acrylic acid or methacrylic acid copolymers, and metal ions can be mentioned. In addition, polyolefins, styrene-based elastomers, polyester-based thermoplastic elastomers, silicone-based thermoplastic elastomers, silicone resins, etc. can also be used. Furthermore, silicone rubber, soft vinyl chloride resins, polybutadiene resins, ethylene-vinyl acetate copolymers, chlorinated polyethylene resins, polyurethane-based thermoplastic elastomers, polyester-based thermoplastic elastomers, silicone-based thermoplastic elastomers, styrene-based elastomers, such as SBS (styrene-butadiene-styrene), SIS (styrene-isoprene-styrene), SEBS (styrene-ethylene-butylene-styrene), SEPS (styrene-ethylene-propylene-styrene), polyolefin resins, fluorine-based resins, etc. may also be used.

In the genetic testing container member of this embodiment, the gene amplification reagent contains at least a nucleic acid synthesizing enzyme.
In this way, a gene amplification reagent containing a nucleic acid synthesis enzyme is mixed with a resin composition and applied onto a substrate, and then PCR is performed using a tube formed using the resulting genetic testing container component and with the mixture fixed inside (inside the container).This enables the user to omit one preparation step for preparing the enzyme in the process of preparing the PCR reaction solution.
As the nucleic acid synthesizing enzyme, a DNA polymerase capable of amplifying DNA or an RNA polymerase capable of amplifying RNA can be used, and for example, Takara ExTaq or the like can be suitably used.

The gene amplification reagent preferably contains at least one selected from the group consisting of a nucleic acid synthesis substrate, a primer, and a buffer solution, together with the nucleic acid synthesis enzyme.
That is, by including two types of reagents in the gene amplification reagent, the user can omit two preparations in the PCR reaction solution preparation process, by including three types of reagents in the gene amplification reagent, the user can omit three preparations, and by including four types of reagents in the gene amplification reagent, the user can omit four preparations.

As nucleic acid synthesis substrates, for example, when DNA is the target, four types of deoxymononucleotide phosphates, dCTP, dATP, dTTP, and dGTP, can be used, and when RNA is the target, four types of deoxymononucleotide phosphates, dCTP, dATP, dUTP, and dGTP, can be used.
As the primers, one or more primer sets consisting of a forward primer and a reverse primer for amplifying the nucleic acid to be tested can be used. In addition, as the buffer solution, a commonly used one can be used.

In addition, in the genetic testing container member of this embodiment, the resin composition is preferably a photosensitive resin composition, and the surface of the mixture is preferably cured by irradiation with ultraviolet (UV) rays.
By hardening the surface of the mixture of the gene amplification reagent and the resin composition in this manner, it becomes possible to stably use the nucleic acid synthesizing enzyme and the like contained in the mixture in PCR.

As the photosensitive resin composition, it is preferable to use one containing a photosensitive resin and a polymerization initiator. As the photosensitive resin, a photocrosslinking type or photopolymerization type photosensitive resin is used, and specifically, it is preferable to use at least one selected from the group consisting of polyvinyl alcohol/stilbazolium-based photosensitive resin, polyvinyl alcohol/vinyl acetate copolymer and acetal compound-based photosensitive resin, photocrosslinking type photosensitive resin such as cinnamic acid or chalcone, photocrosslinking agent (e.g., bisazide compound, diazonium salt, etc.) or a combination with a vinyl polymer copolymer, or photopolymerization type photosensitive resin having an acrylic acid group at the end. In addition, as the polymerization initiator, a photopolymerization initiator, a photoradical generator, a photoacid generator, a photobase generator, etc. can be suitably used.

The genetic testing container of the present invention is characterized in that it is formed using a member for a genetic testing container and a mixture is fixed inside the container.
In this embodiment, the genetic testing container can also be obtained by first manufacturing the container, and then injecting the gene amplification reagent and the resin composition into the container to create a mixture. In this case, the mixture in the genetic testing container can be irradiated with ultraviolet light to harden the surface of the mixture, thereby obtaining a genetic testing container fixed in the container.

The member for a genetic testing container of this embodiment can be produced, for example, as follows.
First, the nucleic acid synthesis enzyme, the nucleic acid synthesis substrate, the primer solution, the buffer solution, and the photosensitive resin composition are mixed using a pipette or a micropipette with a disposable tip or the like attached, and the mixture obtained is applied onto a substrate. The substrate may be made of the same material as that of the above-mentioned genetic testing container member.

Next, the substrate on which the mixture has been applied is preferably dried for a certain period of time (e.g., one hour) at room temperature and normal pressure, and then the mixture is irradiated with ultraviolet light. The ultraviolet light irradiation is preferably performed for two minutes at 25°C, for example.
In this way, a member for a genetic testing container can be obtained in which the surface of the applied mixture is hardened by ultraviolet irradiation.

Furthermore, by molding the obtained member for a genetic testing vessel by blow molding, vacuum molding, pressure molding or the like, a genetic testing vessel in which the mixture is fixed inside the vessel can be obtained.
As described above, it is also possible to obtain a genetic testing container by first manufacturing the container, then injecting a gene amplification reagent and a resin composition into the container to create a mixture, drying the container for a certain period of time as described above, and then irradiating the mixture with ultraviolet light.

Next, how the gene amplification reagent works when PCR is carried out using the gene testing container thus obtained will be described.
PCR can suitably amplify a target region in genomic DNA, for example, by performing the PCR under the following reaction conditions.
(a) 94°C for 10 minutes, (b) 98°C (DNA denaturation step) for 10 seconds, (c) 60°C (annealing step) for 30 seconds, (d) 72°C (DNA synthesis step) for 1 minute (40 cycles of (b) to (d)), (e) 72°C for 10 minutes.

In other words, the genetic testing container is exposed to high temperatures of 90°C or higher during the PCR reaction process, causing the resin composition of the mixture fixed within the container to dissolve and the gene amplification reagents contained within the mixture to become liberated into the genetic testing container.
Thereafter, the target nucleic acid is amplified to produce an amplified product in the same manner as in the case of carrying out a general method for preparing a reaction solution for PCR.

As described above, according to this embodiment, a gene amplification reagent and a resin composition are mixed, and a genetic testing container having a hardened mixture fixed on the surface can be obtained.
By using such a genetic testing container, it is possible to simplify the process of preparing a reaction solution for amplifying nucleic acid without requiring special equipment or facilities.

Tests carried out using the genetic testing container according to the embodiment of the present invention will be described below.
Specifically, PCR was performed using a tube in which a mixture of a genetic testing reagent and a photosensitive resin was hardened by exposure to ultraviolet light and fixed inside, and it was verified whether an amplified product was obtained.

The following strains were used as control bacteria:
Listeria monocytogenes ATCC 15313

This strain was cultured in a Brain Heart Infusion (Difco) at 37° C. for 1 to 2 days.
Next, genomic DNA was extracted from the obtained enrichment medium using RBC Genomic DNA Extraction Kit Mini (RBC Bioscience) according to the manufacturer's instructions.

Four types of samples were prepared as shown in the table below.

Sample 1 was prepared by a general method for preparing a reaction solution for PCR, and the enzyme, primer, substrate, and buffer solution were added during preparation.
Sample 2 uses a genetic testing container in which a mixture of only the enzyme of the genetic testing reagent and a photosensitive resin is immobilized, making it possible to omit the preparation of the enzyme alone.

Sample 3 uses a genetic testing container in which the enzymes, primers, substrates, and a mixture of buffer solution and photosensitive resin of the genetic testing reagents are fixed, making it possible to omit the preparation of these four types of genetic testing reagents.
Sample N was a negative sample, and was prepared using the same genetic testing container as sample 3, but without adding the target nucleic acid.

Specifically, the genetic testing container of Sample 2 was prepared as follows.
0.25 μL of photosensitive resin (BIOSURFINE-AWP-MRH, manufactured by Toyo Gosei Co., Ltd.) and 0.25 μL of Takara Ex Taq HS were mixed in a 0.2 mL PCR tube. After storing the tube in a closed state at 37° C. for 1 hour, the tube was opened to harden the photosensitive resin, and the inside of the tube was irradiated with a UV lamp (light box for photosensitive substrates, manufactured by Sanhayato Co., Ltd.) at 25° C. for 2 minutes to obtain a genetic testing container for sample 2.

The genetic testing container of Sample 3 was prepared as follows.
11.25 μL of photosensitive resin (BIOSURFINE-AWP-MRH, manufactured by Toyo Gosei Co., Ltd.), 0.25 μL of Takara Ex Taq HS, 5.0 μL of 10×EX Taq Buffer, 4.0 μL of dNTP mixer, and 1.0 μL of each primer solution were mixed in a 0.2 mL PCR tube. After storing the tube at 37°C for 1 hour with the tube closed, the tube was opened to harden the photosensitive resin, and the inside of the tube was irradiated with a UV lamp (light box for photosensitive substrates, manufactured by Sanhayato Co., Ltd.) at 25°C for 2 minutes to obtain a genetic testing container for sample 3.

The PCR reaction solution used contained a primer set consisting of a forward primer with the base sequence shown in SEQ ID NO:1 and a reverse primer with the base sequence shown in SEQ ID NO:2 for amplifying the DNA of Listeria. These primers were synthesized by Sigma-Aldrich Japan LLC.

As a PCR reaction solution for sample 1, 50 μl of the following composition was prepared.
1. 10xEx Taq buffer (Mg2+ plus) (20 mM) (Takara Bio) 5.0 μl
2. dNTP mixer 2.5mM each (Takara Bio) 4.0μl
3. 10 μM forward primer (SEQ ID NO: 1) 1.0 μl
4. 10 μM reverse primer (SEQ ID NO: 2) 1.0 μl
5. Takara Ex Taq HS (5U/μl) (Takara Bio) 0.25μl
6. Template DNA (1 ng/PCR reaction solution) 2.0 μl
7. Sterile water (add water until the total volume is 50.0 μl)

The PCR reaction solution for sample 2 was prepared in a total volume of 50 μl (excluding the amount of photosensitive resin) containing the following composition and 0.25 μl of Takara Ex Taq HS that had been hardened in advance in a tube.
1. 10xEx Taq buffer (Mg2+ plus) (20 mM) (Takara Bio) 5.0 μl
2. dNTP mixer 2.5mM each (Takara Bio) 4.0μl
3. 10 μM forward primer (SEQ ID NO: 1) 1.0 μl
4. 10 μM reverse primer (SEQ ID NO: 2) 1.0 μl
5. Template DNA (1 ng/PCR reaction solution) 2.0 μl
6. Sterile water (add water until the total volume is 50.0 μl)

The PCR reaction solution for sample 3 was prepared in a total volume of 50 μl (excluding the amount of photosensitive resin) containing the following composition: 0.25 μl of Takara Ex Taq HS that had been hardened in advance in a tube, 5.0 μl of 10x Ex Taq buffer (Mg2+ plus), 4.0 μl of dNTP mixer, and 1.0 μl each of primer solution.
1. Template DNA (1 ng/PCR reaction solution) 2.0 μl
2. Sterile water (add water until the total volume is 50.0 μl)
The PCR reaction solution for sample N was sterile water (50.0 μl).

Using each of the above PCR reaction solutions, DNA amplification was carried out using a nucleic acid amplifier (Master cycler ep, Eppendorf) under the following conditions.
(a) 94°C 10 min (b) 98°C 10 sec (c) 60°C 30 sec (d) 72°C 1 min (40 cycles of (b) to (d))
(e) 72°C 10 minutes

Electrophoresis of the PCR amplified products was performed using an electrophoresis device (MultiNA, Shimadzu Corporation). Since the length of the amplified product when using the above primers was 163 bp, the presence of an amplified product near 163 bp was judged to be positive. The results are shown in Figure 1.

As shown in FIG. 1, when the genetic testing containers of Samples 2 and 3 were used, an amplification product of the target nucleic acid was obtained, similarly to Sample 1.
In this way, it was confirmed that when the genetic testing container of the above embodiment is used, the reaction solution preparation process can be simplified and PCR can be performed suitably without using special equipment or facilities.

The present invention is not limited to the above-described embodiments and examples, and it goes without saying that various modifications and variations are possible within the scope of the present invention.
For example, when other reagents than those used above are used as the genetic testing reagent, appropriate modifications can be made, such as preparing a mixture including those reagents.

The present invention can be suitably used to specifically detect microorganisms such as bacteria in environmental testing, food testing, etc.

Claims (4)

A mixture containing one or more gene amplification reagents containing at least a nucleic acid synthesizing enzyme and a resin composition is applied to at least a part of a substrate,
the gene amplification reagent comprises a nucleic acid synthesis enzyme, a buffer solution, a nucleic acid synthesis substrate, and a primer;
A member for a genetic testing container, characterized in that the nucleic acid synthesis substrate comprises four kinds of deoxymononucleotide phosphates, dCTP, dATP, dTTP, and dGTP, or four kinds of deoxymononucleotide phosphates, dCTP, dATP, dUTP, and dGTP. The member for a genetic testing container according to claim 1, characterized in that the resin composition is a photosensitive resin composition, and the surface of the mixture is cured by irradiation with ultraviolet light. The member for a genetic testing container according to claim 1 or 2, characterized in that the genetic testing is performed using an amplified product by the PCR method. 4. A genetic testing vessel, comprising the member for a genetic testing vessel according to claim 1, and the mixture is fixed in the vessel.